To support its ongoing monitoring program of therapeutic and diagnostic allergen preparations, LIB has initiated a multifaceted study of the relationship between the structure and function of allergens. These include: 1. Physico-chemical identification of allergens. We have initiated studies on the use of HPLC, MALDI-TOF mass-spectroscopy and microarray analyses in the characterization of target allergens. 2. Role of glycosylation in allergen recognition and processing. Recent advances in the bioengineering and cloning of allergens have raised new questions about the immunogenicity of non-glycosylated proteins. Dr. Soldatova is studying the role of glycosylation in the immunogenicity of recombinant bee venom hyaluronidase. This allergen is being studied for allergenic structural determinants in order to modify them, thus producing an allergens with decreased IgE binding activity but preserved ability to stimulate a T-cell immunity. The study of the allergenic epitopes will be performed simultaneously by two approaches: (1) the study of protein surface epitopes, and (2) the study of potential carbohydrate epitopes. The search of protein surface IgE binding epitopes will be conducted using the monoclonal antibodies against hyaluronidase which are able to compete with IgE antibodies. Preliminary data on the importance of carbohydrate chains of allergens were obtained when Dr. Soldatova has performed the expression of Hya in parallel in bacteria and in baculovirus-infected insect cells. The biological activity of two differently produced proteins was compared and revealed the superior enzymatic and IgE binding activity of baculovirus-expressed hyaluronidase in comparison to E.coli-expressed allergen. Based on these data, the role of the glycosylation for the biological activity of allergens seems to be important. The expression of the biologically active hyaluronidase in baculovirus system made it possible to determine the crystal structure of this important allergen (in collaboration with Dr. Housley Basel Biocenter, Switzerland). The seri of active site and glycosylation site mutants for hyaluronidase have been obtained in collaboration with Dr. Housley (Basel Biocenter, Switzerland). We are now expressing these mutants in baculovirus expression system. First results on the characterization of expressed mutants reveal the preserved IgG- and IgE-binding activity of the mutants on the immunoblots. The results of these studies will contribute to our understanding of the role of glycosylation for the biological function of allergens. 3. Enzyme activity as a measure of allergenicity. Current standards require the presence of active hyaluronidase and phospholipase in venom extracts. We have initiated studies to address the issue of the relevance of these activities for allergenicity. 4. Molecular cloning of a new bee venom allergen, acid phosphatase. Dr. Soldatova has cloned the important allergen of honeybee venom, acid phosphatase, and determined 70% of cDNA sequence. A 30% of nucleotide sequence homology was found with Drosophila and human lysosomal acid phosphatases suggesting that honeybee acid phosphatase is potentially membrane protein. Original data from direct protein sequencing of acid phosphatase correlate only in part with the partial sequence of cloned isoform of the allergen suggesting the presence of other isoforms in the honeybee venom. Keeping in mind, that all other cloned genes of honeybee allergens represented a rare copy genes, it will be reasonable to search for other acid phosphatase isoforms using a cDNA library of honeybee and not by RT-PCR method. The panel of available recombinant allergens of honey bee venom will be extended to three major allergens including phospholipase A2, hyaluronidase and acid phosphatase. Each of these key components of bee venom as well as a melittin (a minor allergen) can then be evaluated for inclusion in a diagnostic panel to detect honeybee allergy, and for the immunotherapy of allergic individuals.